Mattia Sturlese

MMsINC: a large-scale chemoinformatics database

MMsINC (http://mms.dsfarm.unipd.it/MMsINC/search) is a database of non-redundant, richly annotated and biomedically relevant chemical structures. A primary goal of MMsINC is to guarantee the highest quality and the uniqueness of each entry. MMsINC then adds value to these entries by including the analysis of crucial chemical properties, such as ionization and tautomerization processes, and the in silico prediction of 24 important molecular properties in the biochemical profile of each structure. MMsINC is consequently a natural input for different chemoinformatics and virtual screening applications. In addition, MMsINC supports various types of queries, including substructure queries and the novel ‘molecular scissoring’ query. MMsINC is interfaced with other primary data collectors, such as PubChem, Protein Data Bank (PDB), the Food and Drug Administration database of approved drugs and ZINC.

SAR and QSAR study on 2-aminothiazole derivatives, modulators of transcriptional repression in Huntington’s disease

REST/NRSF is a multifunctional transcription factor that represses or silences many neuron-specific genes in both neural and non-neural cells by recruitment to its cognate RE1/NRSE regulatory sites. An increase in RE1/NRSE genomic binding is found in Huntingtons disease (HD), resulting in the repression of REST/NRSF regulated gene transcription, among which BDNF, thus representing one of the possible detrimental effectors in HD. Three 2-aminothiazole derivatives were recently identified as potent modulators of the RE1/NRSE silencing activity through a cell-based gene reporter assay. In this study, the structure-activity relationships (SAR) of a library of commercially available 2-aminoisothiazoles diversely substituted at the amino group or at position 4 has been evaluated. A quantitative structure-activity relationship analysis performed using the Phase strategy yielded highly predictive 3D-QSAR pharmacophore model for in silico drug screening.

Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters

Prestin (SLC26A5) is a member of the SLC26/SulP anion transporter family. Its unique quasi-piezoelectric mechanical activity generates fast cellular motility of cochlear outer hair cells, a key process underlying active amplification in the mammalian ear. Despite its established physiological role, it is essentially unknown how prestin can generate mechanical force, since structural information on SLC26/SulP proteins is lacking. Here we derive a structural model of prestin and related transporters by combining homology modelling, MD simulations and cysteine accessibility scanning. Prestin’s transmembrane core region is organized in a 7+7 inverted repeat architecture. The model suggests a central cavity as the substrate-binding site located midway of the anion permeation pathway, which is supported by experimental solute accessibility and mutational analysis. Anion binding to this site also controls the electromotile activity of prestin. The combined structural and functional data provide a framework for understanding electromotility and anion transport by SLC26 transporters.

Dopamine-derived Quinones Affect the Structure of the Redox Sensor DJ-1 through Modifications at Cys-106 and Cys-53

Background: DJ-1, a protein involved in PD, protects neurons by acting as an oxidative stress sensor. Results: Through adduct formation on DJ-1 cysteines, DAQs induce both structural perturbations and uncoupling of the sensor function. Conclusion: Cys-53 is the most reactive, but Cys-106 modification induces the most severe effects. Significance: A correlation between DJ-1 DAQ-dependent impairment and the degeneration of dopaminergic neurons observed in PD is suggested. The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.

Iron-sulfur cluster binding by mitochondrial monothiol glutaredoxin-1 of Trypanosoma brucei: molecular basis of iron-sulfur cluster coordination and relevance for parasite infectivity.

AIMS Monothiol glutaredoxins (1-C-Grxs) are small proteins linked to the cellular iron and redox metabolism. Trypanosoma brucei brucei, model organism for human African trypanosomiasis, expresses three 1-C-Grxs. 1-C-Grx1 is a highly abundant mitochondrial protein capable to bind an iron-sulfur cluster (ISC) in vitro using glutathione (GSH) as cofactor. We here report on the functional and structural analysis of 1-C-Grx1 in relation to its ISC-binding properties. RESULTS An N-terminal extension unique to 1-C-Grx1 from trypanosomatids affects the oligomeric structure and the ISC-binding capacity of the protein. The active-site Cys104 is essential for ISC binding, and the parasite-specific glutathionylspermidine and trypanothione can replace GSH as the ligands of the ISC. Interestingly, trypanothione forms stable protein-free ISC species that in vitro are incorporated into the dithiol T. brucei 2-C-Grx1, but not 1-C-Grx1. Overexpression of the C104S mutant of 1-C-Grx1 impairs disease progression in a mouse model. The structure of the Grx-domain of 1-C-Grx1 was solved by nuclear magnetic resonance spectroscopy. Despite the fact that several residues--which in other 1-C-Grxs are involved in the noncovalent binding of GSH--are conserved, different physicochemical approaches did not reveal any specific interaction between 1-C-Grx1 and free thiol ligands. INNOVATION Parasite Grxs are able to coordinate an ISC formed with trypanothione, suggesting a new mechanism of ISC binding and a novel function for the parasite-specific dithiol. The first 3D structure and in vivo relevance of a 1-C-Grx from a pathogenic protozoan are reported. CONCLUSION T. brucei 1-C-Grx1 is indispensable for mammalian parasitism and utilizes a new mechanism for ISC binding.

DockBench: An Integrated Informatic Platform Bridging the Gap between the Robust Validation of Docking Protocols and Virtual Screening Simulations

Virtual screening (VS) is a computational methodology that streamlines the drug discovery process by reducing costs and required resources through the in silico identification of potential drug candidates. Structure-based VS (SBVS) exploits knowledge about the three-dimensional (3D) structure of protein targets and uses the docking methodology as search engine for novel hits. The success of a SBVS campaign strongly depends upon the accuracy of the docking protocol used to select the candidates from large chemical libraries. The identification of suitable protocols is therefore a crucial step in the setup of SBVS experiments. Carrying out extensive benchmark studies, however, is usually a tangled task that requires users’ proficiency in handling different file formats and philosophies at the basis of the plethora of existing software packages. We present here DockBench 1.0, a platform available free of charge that eases the pipeline by automating the entire procedure, from docking benchmark to VS setups. In its current implementation, DockBench 1.0 handles seven docking software packages and offers the possibility to test up to seventeen different protocols. The main features of our platform are presented here and the results of the benchmark study of human Checkpoint kinase 1 (hChk1) are discussed as validation test.

Deciphering the Complexity of Ligand-Protein Recognition Pathways Using Supervised Molecular Dynamics (SuMD) Simulations.

Molecular recognition is a crucial issue when aiming to interpret the mechanism of known active substances as well as to develop novel active candidates. Unfortunately, simulating the binding process is still a challenging task because it requires classical MD experiments in a long microsecond time scale that are affordable only with a high-level computational capacity. In order to overcome this limiting factor, we have recently implemented an alternative MD approach, named supervised molecular dynamics (SuMD), and successfully applied it to G protein-coupled receptors (GPCRs). SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand, and also from its receptor binding affinity. In this article, we present an extension of the SuMD application domain including different types of proteins in comparison with GPCRs. In particular, we have deeply analyzed the ligand-protein recognition pathways of six different case studies that we grouped into two different classes: globular and membrane proteins. Moreover, we introduce the SuMD-Analyzer tool that we have specifically implemented to help the user in the analysis of the SuMD trajectories. Finally, we emphasize the limit of the SuMD applicability domain as well as its strengths in analyzing the complexity of ligand-protein recognition pathways.

Side chain cyclization based on serine residues: synthesis, structure, and activity of a novel cyclic analogue of the parathyroid hormone fragment 1-11.

The N-terminal region of the parathyroid hormone (PTH) is sufficient to activate the G-protein-coupled PTH receptor 1 (PTHR1). The shortest PTH analogue displaying nanomolar potency is the undecapeptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) that contains two helix-stabilizing residues (Aib(1,3)). To increase the helical character and proteolytic stability of this linear peptide, we replaced Gln(6,10) with (a) Lys(6) and Glu(10) to introduce a lactam bridge and (b) Ser(6,10) to form a diester bridge upon cross-linking with adipic acid. These cyclopeptides were, respectively, 468-fold less and 12-fold more potent agonists than the linear analogue. Despite their different potencies, all three analogues adopted similar α-helix structures, as shown by NMR and molecular dynamics studies. However, the diester bridge could better mimic the orientation and chemical properties of the side chains of Gln(6) and Gln(10) in the linear PTH analogue than the lactam moiety. This is apparently important for efficient receptor activation and provides further insights into the receptor-bound ligand conformation.

DockBench as docking selector tool: the lesson learned from D3R Grand Challenge 2015

Structure-based drug design (SBDD) has matured within the last two decades as a valuable tool for the optimization of low molecular weight lead compounds to highly potent drugs. The key step in SBDD requires knowledge of the three-dimensional structure of the target-ligand complex, which is usually determined by X-ray crystallography. In the absence of structural information for the complex, SBDD relies on the generation of plausible molecular docking models. However, molecular docking protocols suffer from inaccuracies in the description of the interaction energies between the ligand and the target molecule, and often fail in the prediction of the correct binding mode. In this context, the appropriate selection of the most accurate docking protocol is absolutely relevant for the final molecular docking result, even if addressing this point is absolutely not a trivial task. D3R Grand Challenge 2015 has represented a precious opportunity to test the performance of DockBench, an integrate informatics platform to automatically compare RMDS-based molecular docking performances of different docking/scoring methods. The overall performance resulted in the blind prediction are encouraging in particular for the pose prediction task, in which several complex were predicted with a sufficient accuracy for medicinal chemistry purposes.

Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents

A small library of 7-pyrrolo[3,2-f]quinolinones was obtained by introducing benzoyl, sulfonyl and carbamoyl side chains at the 3-N position, and their cytotoxicity against a panel of leukemic and solid tumor cell lines was evaluated. Most of them showed high antiproliferative activity with GI50s ranging from micro-to sub-nanomolar values, and these values correlated well with the inhibitory activities of the compounds against tubulin polymerization. Based on a recently proposed colchicine bind site inhibitors (CBSIs) pharmacophore, the interactions of the novel 7-PPyQs at the colchicine domain were rationalized. The most active compounds (4a and 4b) did not induce significant cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. In particular, 4a was a potent inducer of apoptosis in both the HeLa and Jurkat cell lines. On the other hand, the sulfonyl derivative 4b exhibited a lower potency in comparison with 4a. With both compounds, induction of apoptosis was associated with dissipation of the mitochondrial transmembrane potential and production of reactive oxygen species, suggesting that cells treated with the compounds followed the intrinsic pathway of apoptosis.